Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

Momochika Kumagai,Akira Yabuki,Mika Otsuka,Takahisa Hamano,Sanae Kato,Nanami Arakawa,Osamu Yamato,Nobuyuki Kashiwagi

doi:10.3390/separations8080128

Abstract

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

Highlights

Carnosine (CAR) and its methylated analogs, anserine (ANS) and balenine (BAL), are representative histidine-containing dipeptides with an imidazole ring skeleton (Figure 1).These imidazole dipeptides (IDPs) are found in the skeletal muscles of a wide range of animal species [1]
No significant interfering peaks were observed on the retention time of ANS, CAR, and BAL in the pooled dolphin serum (Figure 3)
The quantification values of IDPs in all dolphin sera analyzed in this study exceeded these Limit of Quantification (LOQ) values. These results suggested that this method was sensitive enough to measure IDPs in dolphin serum

Summary

Introduction

Carnosine (CAR) and its methylated analogs, anserine (ANS) and balenine (BAL), are representative histidine-containing dipeptides with an imidazole ring skeleton (Figure 1). These imidazole dipeptides (IDPs) are found in the skeletal muscles of a wide range of animal species [1]. It has been reported that marine mammals, unlike other mammals, have skeletal muscles rich in BAL [2,3] These compounds appear to have many beneficial effects on physiological functions such as buffering [4], antioxidative activity [5,6,7], and advanced glycation end-product inhibitory activities [8] and, may reflect the health condition of animal species. IDPs may be useful indicators of the health of aquarium dolphins and aimed to develop an analytical method to measure the IDP content in the serum of bottlenose dolphins.

Objectives

Methods

Results

Conclusion